1. Field of the Invention
The present invention relates to modified protein pores, and, more specifically, to bioengineered protein pores that can be utilized as biosensors.
2. Description of the Related Art
Protein-based nanosensors represent an emergent alternative to current analytical devices in biomedical molecular diagnosis, because of the enhanced selectivity, specificity, and versatility of the protein receptor-ligand recognition. In the last decade, significant progress in protein engineering has enabled the design, synthesis, and purification of protein nanopores customized to execute numerous complex tasks. Currently, the engineering of biological nanopores is focused on pore-forming toxins and bacterial outer membrane proteins, because their robust β-barrel structure makes them the convenient choice for developing sensing technologies. The major benefits of using protein nanopores include knowledge of their accurate structure at atomic resolution, the ability to implement functional groups at strategic positions within their interior, and great prospects for parallelization and integration into nanofluidic devices. Despite these advantageous features, one persistent limitation is the lack of a methodology for preparing stiff protein scaffolds that maintain their functionality under a wide spectrum of environmental conditions. Moreover, these protein nanopores are multimeric, a trait that causes their targeted design to be a tedious and laborious process, because of the numerous permutations and combinations of engineered and native subunits within the same heteromeric protein.
The protein which has served as a benchmark and the archetype for the engineering of proteins nanopores is the staphylococcal endotoxin α-hemolysin (“αHL”). While this protein has been the mainstay of nanopore engineering, it does have two major limitations. First, it has a narrow constriction point with a diameter of 1.5 nm, therefore limiting this pore to the detection of small chemicals and analytes which are less than 1.5 nm in diameter. Second, the heptameric nature of this protein makes the engineering of the protein difficult. Recently, the former limitation has been overcome by engineering the 3.6 nm-wide phi29 motor protein to serve as channel forming nanopore (2.6 hemolysin diameters (“HD”)), which permits the translocation of dsDNA (˜2 nm). Yet, this newly engineered pore is dodecameric, and, similar to the αHL pore, its stoichiometry still poses a limitation to engineering. Thus, a monomeric β-barrel wider protein pore is still highly desired for protein engineering for future biosensing applications that involve proteins analytes and dsDNA.